Mechanism of phospholipase D activation induced by prostaglandin D₂ in osteoblast-like cells: Function of Ca²⁺/calmodulin

Prostaglandin D₂ (PGD₂) stimulated the formation of choline in a dose-dependent manner in the range between 10 nM and 10 μM. The effect of PGD₂ on the formation of inositol phosphates (EC₅₀ was 20 nM) was more potent than that on the formation of choline (EC₅₀ was 0.5 μM). The formation of choline stimulated by a combination of PGD₂ and 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, was additive. Staurosporine, an inhibitor for protein kinases, enhanced the PGD₂-induced formation of choline, but H-7, another inhibitor for protein kinases, had little effect. PGD₂ stimulated Ca²⁺ influx from extracellular space dose-dependently. The depletion of extracellular Ca²⁺ by EGTA reduced the PGD₂-induced formation of choline. W-7 and trifluoperazine dihydrochloride, antagonists of calmodulin, dose-dependently inhibited the PGD₂-induced choline formation. These results strongly suggest that PGD₂ activates phospholipase D in a Ca²⁺/calmodulin dependent manner in osteoblast-like cells, and that protein kinase C is not essential for the PGD₂-induced activation of phospholipase D.